Field of the Invention
The present disclosure generally relates to a protein engineering and expression platform that uses a protease to process a precursor polypeptide into individual subunit proteins and which minimizes protease-associated damage to the host cells used to recombinantly-express the precursor protein. One example of this platform comprises an engineered foot-and-mouth disease virus (“FMDV”) P1 precursor polypeptide, which has been engineered to contain Tobacco Etch Virus NIa protease (“TEV protease”) sites at the junctions defining separate viral structural proteins in the unprocessed precursor polypeptide, and a Tobacco Etch Virus NIa protease that recognizes the sites engineered into the FMDV P1 precursor polypeptide. FMDV P1 precursor polypeptide is conventionally processed by the FMDV 3C protease which is toxic to many host cells. The invention obviates the need for use of a toxic FMDV 3C protease for production of processed FMDV structural proteins from the P1 precursor and thus provides a more efficient and productive platform for the recombinant production of FMDV capsid proteins for use in vaccines and/or diagnostic tools for foot-and-mouth disease (FMD).
Description of Related Art
The “background” description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description which may not otherwise qualify as prior art at the time of filing, are neither expressly or impliedly admitted as prior art against the present disclosure.
FMDV, a prototypic aphthovirus within the Picornaviridae family, is the causative agent of a highly infectious and sometimes fatal disease, FMD, that affects cloven-hoofed animals such as cattle, pigs, sheep, goats, deer and other animals with divided hooves. There are seven major antigenically distinct FMDV serotypes (A, O, C, Asia 1 and South African Territories or SAT 1, 2 and 3) with multiple subtypes or topotypes existing within each serotype. Infection with any one serotype does not confer protective immunity against another. Serotype O is the most common serotype worldwide.
After an animal is infected with FMDV, the first signs of illness usually appear within 2 to 14 days. These usually include high fever for 2-3 days followed by blisters inside the mouth and on the feet that that may rupture and cause lameness.
FMD outbreaks cause significant agro-economic losses and have severe implications for animal farming throughout much of the world. For example, the estimated costs attributable to the 2001 outbreak of FMD in the U.K. were £8 billion, including the costs of slaughtering and sanitarily disposing of 6 million livestock. The virus causing the disease is highly contagious and can be spread by direct contact, through aerosols to uninfected livestock by infected livestock, or by domestic or wild animals. FMDV may be also transmitted by contact with contaminated farming equipment, vehicles, clothing, or feed. Consequently, the containment of FMDV demands considerable effort and expenses including those required for vaccination, vigilance and strict monitoring of livestock, or culling and disposal of infected livestock, as well as for accommodating transport and trade restrictions, quarantines and other administrative and legal issues.
The current most commonly used FMD vaccines utilize whole virus that has been killed, inactivated, and/or attenuated. A whole virus vaccine includes dozens of FMDV antigens to provide a broad spectrum of immunity against different. FMDV strains and variants, including those arising due to antigenic drift or antigenic shift. However, this vaccine platform is fraught with many limitations and shortcomings. Animals immunized with the whole virus are difficult to distinguish from infected animals. Once exposed to the whole virus antigens one's ability to immunologically differentiate between infected and vaccinated animals is limited. The efficacy of the current vaccine formulations is also limited by immunogenic instability and short vaccine shelf life that results in a loss of potency upon transportation or storage that results in subsequent induction of insufficient immunity or immunity of a short duration. Furthermore, the set-up and running costs of producing the current FMDV vaccine in potent form and securing and maintaining its production facilities are very high. For example, the mode of producing current FMDV vaccine raises safety concerns due to the possibility of virus escape from vaccine production facilities. Recombinant production of FMDV antigens, which could avoid the problems inherent to use of whole virus-based vaccines, is impeded by the promiscuous proteolytic activity of the FMDV 3C protease which is required for the processing of FMDV antigens from the FMDV P1 precursor polypeptide, but which also cleaves proteins in host cells used to express recombinant FMDV antigens. Native FMDV 3C protease is toxic to host cells and significantly reduces their ability to express immunogenic FMDV antigens in significant amounts.
NIa proteases, such as TEV protease, were known but had never been tested or applied in a platform for the production of FMDV antigens or immunogens. It was unknown whether such proteases would ameliorate or exacerbate the problems associated with the use of FMDV 3C protease or other picornavirus proteases. Similarly, the effects of omission of expression of a FMDV 3C protease on the recombinant production and subsequent assembly of FMDV structural proteins into FMDV capsids had never been investigated.
There is a continuing need and interest in the development of new second or third generation vaccines that safely generate strong, stable, and broad protective immune responses against FMDV. With these objectives in mind, the inventors developed modifications to FMDV P1 precursor polypeptide to permit it to be cleaved and processed by a potyvirus NIa protease, such as TEV protease, thus producing immunogenic viral antigens, while reducing or eliminating the cytotoxic effects produced by the FMDV 3C protease conventionally used to recombinantly produce FMDV antigens and immunogens.